p53 has clearly been implicated as a tumor suppressor in human cancer with mutation of the p53 gene being a common event. p53 has a well-characterized role in mediating the cellular response to DNA damage. The checkpoint in the G1 phase has been shown to be strictly p53-dependent. Due to the existence of a G2/M checkpoint that occurs in its absence, the precise role of p53 in preventing mitotic entry has been elusive. The studies in this application will address this key aspect of p53 biology. Three specific aims are proposed to determine the molecular details of p53 function in this checkpoint in response to transient or sustained DNA damage. In the first aim, p53-dependent mechanisms of transcriptional regulation that affect mitotic entry and progression will be elucidated. The genes encoding mitotic regulators including Cdc25C, Survivin, Cdc20, and Cyclin B1 are targets for transcriptional downregulation. To elucidate the molecular basis, the regulation of the cdc25C gene will be studied as being representative. Insights gained in the detailed study of Cdc25C repression will then be examined for p53-dependent transcriptional downregulation of Survivin, Cdc20, and Cyclin B1. In the second aim, p53-dependent mechanisms of post-transcriptional regulation in response to DNA damage will be studied. These include novel roles for Mdm2 in affecting the ability of p21CIP1 to inhibit cyclin-dependent kinases and in regulating Cdc25C protein stability. Preliminary data show that protein stability of not only Cdc25C, but also Survivin, Cdc20, and Cyclin B1 is regulated by p53. This intriguing finding will be further explored. In the third aim, the role of p53 and its downregulated targets in mitotic entry and progression after transient or sustained DNA damage will be examined. Upon transient treatment with DNA damaging agents wild-type p53 cells reversibly arrest and repair the damage, whereas p53 null cells fail to do so and die. The molecular basis for these effects will be elucidated. The proposed studies address a central issue in understanding p53 function, namely how it mediates cell cycle checkpoints in response to DNA damage. The innovation of these planned experiments include the study of a new target for p53 that involves transcriptional repression occurring in a DNA binding-dependent manner, the novel observation that p53 regulates protein stability of particular mitotic regulators as part of checkpoint activation, and the intriguing possibility that Mdm2 plays a positive role as a downstream effector of p53-mediated cellular responses. The significance of this research relates to the clinical implications of selective targeting of tumor cells with a defective p53 pathway, especially given the frequency of p53 mutation in cancer. Taken together, the planned experiments will elaborate a detailed understanding of how p53 mediates DNA damage checkpoints. This is expected to provide new avenues of pursuit that are relevant for prognosis and treatment of human cancer.